This invention pertains to thermolabile phosphate protecting groups, intermediates therefor and methods of using them in oligonucleotide synthesis.
There are significant potential therapeutic applications for oligonucleotides. The therapeutic application of oligonucleotides is based on the selective formation of hybrids between antisense oligonucleotides and complementary nucleic acids, such as messenger RNAs (mRNAs). Such hybrids inhibit gene expression by preventing protein translation. Nuclease-resistant oligonucleotides are highly desirable in this regard. Nucleosides bearing phosphorothioate internucleotide linkages are well-known for such nuclease resistance and, thus, are undergoing rapid development.
In view of their significant potential therapeutic application, there is a high demand for improved methods of preparing oligonucleotides and analogues thereof. A number of methods for synthesizing oligonucleotides have been developed. The most commonly used synthetic method for the synthesis of thioated oligonucleotides is the phosphoramidite method with stepwise sulfurization (see, e.g., U.S. Pat. Nos. 4,415,732, 4,668,777, 4,973,679, 4,845,205, and 5,525,719). Essentially, a phosphate precursor is sulfurized such that a sulfur atom is substituted for one of the non-bridging oxygen atoms normally present in phosphodiesters. This method uses tricoordinated phosphorus precursors that normally produce products containing a mixture of different thioated oligonucleotide stereoisomers, primarily due to the use of non-stereoselective and non-stereospecific acid-catalyzed nucleophilic substitution reactions.
Protecting groups for internucleosidic phosphorus linkages and associated deprotection methods are well-known in the art, and have been described, for example, in U.S. Pat. Nos. 4,417,046, 5,705,621, 5,571,902 and 5,959,099. However, the methods presently used for removing internucleosidic phosphorus protecting groups are disadvantageous in that they employ harsh reagents, such as bases (e.g., ammonium hydroxide) and acids (e.g., trichloroacetic acid). Under these deprotection conditions, there is a greater risk of problems, such as by-product formation and degradation of the desired oligonucleotide, which make oligonucleotide purification more difficult and increase the overall cost, particularly in large-scale production processes. Moreover, the range of structural analogs that one can prepare is limited to those that are stable under the acidic and/or basic deprotection conditions that are commonly employed in the art.
Accordingly, there is a need for internucleosidic phosphorus protecting groups that can be removed under milder conditions and methods of making and using such protecting groups. Removal of such protecting groups should be fast and should be carried out under conditions that minimize the possibility for degradation of the desired oligonucleotide. In addition, the intermediates that introduce such protecting groups should be easy to synthesize inexpensively on a large scale. It is, therefore, of prime importance to develop low-cost, protected intermediates for oligonucleotide synthesis which are easy to synthesize, couple efficiently during stepwise synthesis, and are deprotected quickly in high yield under mild conditions.
The invention provides such protecting groups and methods. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The invention provides a method of thermally deprotecting an oligonucleotide. The method comprises heating an oligonucleotide of the formula: 
in a fluid medium, at a substantially neutral pH, at a temperature up to about 100xc2x0 C. to produce an oligonucleotide of the formula: 
wherein R is a thermolabile protecting group of the formula: 
R1 is H, R1a, OR1a, SR1a or NR1aR1axe2x80x2, wherein R1a and R1axe2x80x2 are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, when R1 is NR1aR1axe2x80x2, R1a and R1axe2x80x2, together with the nitrogen atom to which they are bonded, comprise a heterocycle. Substituent X1 is O, S or Se and substituent X is O or S. Substituent Z is O, S, NR2a, CR2aR2axe2x80x2 or CR2aR2axe2x80x2CR2bR2bxe2x80x2, wherein R2a, R2axe2x80x2, R2b and R2bxe2x80x2 are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R1a or R1axe2x80x2, in combination with any of R2a, R2axe2x80x2, R2b or R2bxe2x80x2, together with Cxe2x95x90X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof. R1 is not R1a when Z is S, Z is not CR2aR2axe2x80x2 or CR2aR2axe2x80x2CR2bR2bxe2x80x2 when R1 is SR1a, and Z is not O or S when R1 is H.
Substituents R2, R2xe2x80x2, R3 and R3xe2x80x2 are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R2 or R2xe2x80x2, in combination with R3 or R3xe2x80x2, together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formulae: 
wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen, provided that the aromatic ring, which bears substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa).
Substituents R1, R2a, R2b, R2bxe2x80x2, R2, R2xe2x80x2, R3 or R3xe2x80x2 can be unsubstituted substituted, as further described herein. Substituents R4 and R15 are the same or different and each is H, a hydroxyl protecting group, or a solid support.
Q and Q1 are the same or different and each is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide. Variable n represents an integer from 1 to about 300. When n is greater than 1, each Q is independently selected.
The present invention further provides a novel compound selected from the group consisting of compounds of the formulae: 
wherein R is a thermolabile protecting group as defined herein, R4, R15 and X1 are as defined herein, and W is a dialkylamino group.
The present invention further provides method of producing an oligonucleotide. The method comprises:
(a) reacting a nucleophile of the formula:
R4xe2x80x94Oxe2x80x94Q1xe2x80x94OH;
with an electrophile of the formula: 
wherein R, R4, Q, Q1 and W are as defined herein, and R15 is a protecting group, under conditions to displace W and produce an adduct comprising a tricoordinated phosphorus atom;
(b) reacting the product obtained in step (a) with a reagent selected from the group consisting of oxidizing agents, sulfurizing agents, and selenizing agents to produce a protected oligonucleotide of the formula: 
wherein n=1;
(c) cleaving R15 from the protected oligonucleotide from step (b) to produce a nucleophile;
(d) optionally repeating steps (a)-(c) until an oligomer of a specified length is obtained; and
(e) thermally deprotecting the thermolabile protecting group R in accordance with the present invention.